Method for isolating a target biological material, capture phase, detecting phase and reagent containing them

ABSTRACT

The invention concerns a method for isolating a target biological material contained in a sample, which consists in providing a capture phase comprising an organic molecule having at least a reactive function and at least a protein material capable of recognizing or binding, specifically and directly or indirectly, with the target biological material, said protein material having a specific covalent binding site with the organic molecule reactive function, consisting of at least a tag comprising at least six contiguous lysine, or lysine derivative residues, the method consists in contacting said target biological material with at least the capture phase; and detecting the target biological material fixed on the capture phase: The invention also concerns the capture and detection phases, and a reagent containing them.

The invention relates to a method for isolating a target biologicalmaterial contained in a sample, using a capture phase comprising anorganic molecule containing at least one reactive function, and at leastone protein material capable of recognizing or binding, specifically anddirectly or indirectly, to the target biological material, said proteinmaterial containing a specific site for covalently binding to thereactive function of the organic molecule.

A method for immobilizing a protein on a solid support, by means ofreactive functions, in order to stimulate the proliferation or growth ofT cells and the production of killer lymphocytes is known from documentEP-A-0,319,012. To this end, the protein has, at its C-terminal end, acovalent-binding site for binding to said reactive functions, whichconsists of an amino acid sequence in which are distributed two to fourlysine residues.

The problem posed by the covalent-binding site of this prior art lies inthe fact that, in a diagnostic application, the results of the isolationof a target biological material with which it is capable of binding aresimilar to those obtained with a protein material which does not containsuch a binding site.

According to the invention, a method is provided for isolating a targetbiological material by using at least one capture phase which comprisesa protein material containing a covalent-binding site which allowsefficient orientation of said protein material, and leads to sensitive,high-quality detection of said biological material.

Thus, the method of the invention, for isolating a target biologicalmaterial contained in a sample, comprises the following steps:

a capture phase is provided, which comprises an organic moleculecontaining at least one reactive function and at least one proteinmaterial capable of recognizing or binding, specifically and directly orindirectly, to the target biological material, said protein materialcontaining a specific site for covalent binding to the reactive functionof the organic molecule, which consists of at least one tag comprisingat least six contiguous lysine or lysine-based residues,

said target biological material is placed in contact with at least onecapture phase, and

the target biological material bound to the capture phase is detected.

According to the invention, the expression “isolating a biologicalmaterial” means the binding, separation, isolation, detection and/orquantification of this material, the enrichment of a fraction withtarget biological material, according to a qualitative and/orquantitative, specific or aspecific binding method.

According to one variant of the method, the capture phase can alsocomprise a label, and, in this case in particular, it can consist of adetection phase.

According to another variant of the method, a detection phase is alsoprovided, which comprises an organic molecule containing at least onereactive function, at least one protein material capable of recognizingor binding, specifically and directly or indirectly, to said targetbiological material, and a label, said protein material containing aspecific site for covalent binding to the reactive function of theorganic molecule, which consists of at least one tag comprising at leastsix contiguous lysine or lysine-based residues.

In this case, the organic molecules in the capture phase and in thedetection phase, respectively, can be identical or different, and theprotein materials in the capture phase and in the detection phase,respectively, can be identical or different.

As is understood by the invention, a sample comprises any sample capableof containing a biological material, in particular a sample such as thatobtained from—a biological fluid, a sample of food origin, or a cellculture.

The sample consists of all or part of another sample: in particular itcan consist of an aliquot or a dilution.

A protein material according to the invention comprises proteins, inparticular recombinant proteins, especially antigens, antibodies andpeptides such as synthetic peptides. The method of the invention mayalso be carried out with a material such as peptide analogues of nucleicacids (PNA).

The term “organic molecule” means a molecule of variable size; thus,this refers equally to a small molecule such as a hapten and to amacromolecule such as a polymer.

As examples of haptens, mention may be made of a hormone, a vitamin,such as biotin, or a medicinal product. In this case, the method of theinvention can comprise, before the step of detecting the targetbiological material, a step of binding the organic molecule to a carriermolecule. Preferably, the hapten is biotin and the carrier molecule isavidin.

A polymer as used according to the invention is a polymer in particulateor in linear form. It can be a homopolymer chosen in particular frompolylysine and polytyrosine, or a copolymer chosen in particular frommaleic anhydride copolymers, N-vinylpyrrolidone copolymers, natural orsynthetic polysaccharides, polynucleotides and amino acid copolymerssuch as enzymes. Advantageously, it is a copolymer chosen from maleicanhydride/methyl vinyl ether copolymer,N-vinylpyrrolidone/N-acryloxysuccinimide copolymer, poly-6-aminoglucose,and enzymes such as horseradish peroxidase (HRP) or alkaline phosphataseor derivatives thereof bearing at least one reactive function.

The expression “reactive function of the organic molecule” means eithera reactive function chosen in particular from ester, halocarbonyl,sulfhydryl, disulfide, epoxide, haloalkyl and aldehyde functions; or afunction which can be activated by an activating agent such ascarbodiimides or homo- or heterobifunctional compounds. By way ofexample, an activatable function is chosen in particular from acid,amine and hydroxyl functions.

The covalent-binding sites defined above can exist naturally in theprotein material. Alternatively, they can be “incorporated” beforehandinto the protein material, in the form of a tag, according to techniqueswhich are well known to those skilled in the art, such as the techniqueused to purify proteins by the IMAC (immobilized metal ion-affinitychromatography) method on resins (1, 2). By way of example, such sitescan be incorporated into a protein material, and in particular aprotein, by genetic engineering in order to obtain recombinant proteins.

A tag can be defined as an amino acid sequence which is incorporatedinto, i.e. added to, the original structure of the protein material,which is introduced into a preferred place in said original structure inorder to allow it to be exposed in a relevant manner, in particular withregard to its covalent binding to the organic molecule.

In accordance with the invention, a covalent-binding site of the proteinmaterial considered can consist of a tag comprising six or more lysineor lysine-based residues, and optionally other amino acids, or canconsist of several of said tags.

The term “lysine-based” means that the lysine can be chemicallymodified, provided that these modifications essentially preserve or evenenhance the specificity of the covalent-binding site. Examples which maybe mentioned are the replacement of L-lysine with D-lysine, andvice-versa; a modification of the lysine side chain, such as anacetylation of the amine function or an esterification of the carboxylfunction; a modification of the peptide bonds such as, for example,carba, retro, inverso, retro-inverso, reduced and methylenoxy bonds.

The tag(s) described above can be found in any place in the primarystructure of the protein material. Preferably, it is located at the N-or C-terminal end of the protein material.

According to the present invention, various methodes are also definedfor isolating a target biological material contained in a sample,depending, in particular, on its nature:

if the target biological material is an antibody, the protein materialcomprises an antigen which specifically recognizes said antigen;

if the target biological material is an antigen, the protein materialcomprises an antibody which specifically recognizes said antigen.

Other subjects of the invention are outlined below.

Thus, the invention relates to a phase for capturing a target biologicalmaterial which comprises an organic molecule containing at least onereactive function and at least one protein material capable ofrecognizing or binding, specifically and directly or indirectly, to thetarget biological material, said protein material containing a specificsite for covalent binding to the reactive function of the organicmolecule, which consists of at least one tag comprising at least sixcontiguous lysine residues; preferably, the organic molecule is apolymer or a hapten.

An advantageous polymer is particulate or linear and chosen inparticular from homopolymers such as polylysine or polytyrosine; andcopolymers such as maleic anhydride copolymers, N-vinylpyrrolidonecopolymers, natural or synthetic polysaccharides, polynucleotides andamino acid copolymers such as enzymes. Even more advantageously, thepolymer is chosen from maleic anhydride/methyl vinyl ether copolymer,N-vinylpyrrolidone/N-acryloxysuccinimide copolymer, poly-6-aminoglucose,horseradish peroxidase (HRP) and alkaline phosphatase.

If the organic molecule is a hapten, the capture phase can also comprisea suitable carrier molecule. Preferably, the hapten is biotin and, ifnecessary, the carrier molecule is avidin.

Advantageously, the tag as defined above is placed at the N- orC-terminal end of the protein material.

The reactive function of the organic molecule is chosen in particularfrom ester, acid, halocarbonyl, sulfhydryl, disulfide, epoxide,haloalkyl and aldehyde functions.

The invention also relates to a phase for detecting a target biologicalmaterial, having the same characteristics as the above capture phase andalso comprising a label.

As indicated above, the organic molecule can be a polymer or a hapten.According to the second possibility, the organic molecule consisting ofthe hapten also represents the label.

The label for the detection phase is preferably chosen from the groupconsisting of an enzyme, a protein, a peptide, an antibody, a haptensuch as biotin or iminobiotin, a fluorescent compound such as rhodamine,a radioactive compound, a chemiluminescent compound, an electron-densitycomponent, a magnetic component and the like.

Another subject of the invention is a reagent for isolating a targetbiological material, comprising a capture phase of the invention and/ora detection phase of the invention.

Advantageously, the capture phase is bound, directly or indirectly, to asolid support, by passive absorption or by covalency.

The solid support can be in any suitable form, such as a plate, a cone,a bead which is optionally radioactive and/or fluorescent and/ormagnetic and/or conductive, a bar, a tube of glass, a well, a sheet, achip or the like. It is chosen from among polystyrenes,styrene/butadiene copolymers, styrene/butadiene copolymers mixed withpolystyrenes, polypropylenes, polycarbonates, polystyrene/acrylonitrilecopolymers, styrene/methyl methylmethacrylate copolymers, from amongsynthetic and natural fibres, and from among polysaccharides andcellulose derivatives, glass, silicon and derivatives thereof.

As will be demonstrated in the experimental section below, the Authorshave obtained monoclonal antibodies directed against the tags as definedabove and in particular against the tag which consists of a sequence ofsix lysine residues.

It is thus possible to envisage the use of such a tag in a method forisolating a biological material, according to which:

a capture phase is provided, said target biological material is placedin contact with at least the capture phase, and the target biologicalmaterial bound to the capture phase is detected, according to whichmethod the capture phase comprises an organic molecule which comprisesor consists of an anti-tag antibody and at least one protein materialcapable of recognizing or of binding, specifically and directly orindirectly, to the target biological material, said protein materialcontaining a specific site for binding to the organic molecule, whichconsists of at least one tag comprising at least six contiguous lysineor lysine-based residues.

Such a method can also comprise the use of a detection phase, which hasthe characteristics of the capture phase as has just been described, andalso comprising a label. This label can advantageously consist of a saidantibody.

The characteristics and advantages of the various subjects of theinvention are illustrated below, in support of Examples 1 to 6 and FIGS.1 to 7.

FIG. 1 represents a graph reflecting the yield of coupling by covalentbinding between the protein RH24K (light-shaded bars) and the proteinRH24 (dark-shaded bars) and the copolymer, for different couplingconditions; Acet=acetate; Phos=phosphate; Bor=borate; Carb=carbonate.

FIG. 2 represents a graph of comparison between the stability as afunction of time (in days) of the polymer (MAVE)/protein (RH24K)conjugate compound (curve ▪) and that of the free RH24K protein (curve).

FIG. 3 represents a scheme summarizing the “sandwich” detectiontechnique.

FIG. 4 represents a graph reflecting the immunoreactivity of thepolymer/RH24 conjugate compounds (curve ), and polymer/RH24K conjugatecompounds (curve ▪), with respect to a monoclonal antibody.

FIG. 5 represents a graph reflecting the immunoreactivity of thepolymer/RH24 conjugate compounds (curve ) and polymer/RH24K conjugatecompounds (curve ▪), with respect to serum.

FIG. 6 illustrates the results of a sandwich and indirect ELISA testwith the antibody IG2D4, using a graph representing the OD as a functionof the antibody titer.

FIG. 7 illustrates the results of an indirect ELISA test with theantibody IG2D4, using a graph representing the OD as a function of theantibody titer.

EXAMPLE 1 Expression and Purification of RH24K

pMH24 (Cheynet et al., 1993) was modified by insertion of a syntheticadapter at the 3′ end of the p24 gene in order to express RH24K,corresponding to RH24 which has at its C-terminal end a specific sitefor covalent binding referred to as a “polylysine tail” and consistingof 6 contiguous lysine residues.

The adapter was prepared beforehand by hybridization of twooligonucleotides having the following respective sequences:

SEQ ID NO: 1: 5′ GGGAAGAAGA AGAAGAAGAA GTCTGTCGAC GAATCTCT 3′

SEQ ID NO: 2: 5′ CTAGAGAGAT TCGTCGACAG ACTTCTTCTT CTTCTTCTTCC C 3′

in 10 mM Tris, 50 mM NaCl, 1 mM DTE, 10 mM MgCl₂, pH 7.5 buffer, byheating at 70° C. for 5 minutes followed by slow cooling first to 37° C.over 30 minutes and then to room temperature over 30 minutes. Thisadapter was then cloned between the sites SmaI and XbaI of pMH24 at the3′ end of the RH24 gene. The resultant plasmid, pMH24K3′, was amplifiedin the E. coli XLI bacterial strain (recA1, endA1, gyrA96, thi-1,hsdR17, supE44, relA1, lac [F′ proAB, lacIPZΔAM15, Tn10 (tet^(r))] andthen sequenced by the Taq Dye Deoxy Terminator Matrix Standart method(Applied Biosystems). The sequence obtained and the correspondingsequence on the protein obtained at the C-terminal are given below.

           SmaI        5′ATG                          CCC GGG AAG AAGAAG AAG AAG AAG Nter Met . . . [247 amino acids] . . . Pro Gly Lys LysLys Lys Lys Lys                    XbaI TCT GTC GAC GAA TCT CTC TAG A 3′(SEQ ID NO: 3) Ser Val Asp Glu Ser Leu       Cter (SEQ ID NO: 4)

The protein is expressed under the control of the Tac promoter in the E.coli XLI bacterial strain (see above). A preculture of E. coli XLIcontaining the expression vector, is prepared beforehand overnight at37° C. in Luria broth (LB) in the presence of ampicillin (amp; 50 μg/ml)and tetracycline (tet; 12 μg/ml) in order to inoculate the LB-amp-tetmedium (1/40 to 1/25); this medium is cultured until an OD at 600 nm of0.6 is obtained. Expression of the protein is then induced by additionof isopropyl-β-D-thiogalactopyranoside (IPTG) to the medium at aconcentration of 1 mM for 2h30 at 37° C. with stirring. Aftercentrifugation at 5000 revolutions/min (JA21 rotor, Beckman) for 10minutes at 4° C., the bacteria are taken up in a 50 mM Tris-HCl, 1 mMEDTA, 100 mM NaCl, 10 mM MgC12, pH 8 buffer (3 ml/g of biomass) in thepresence of protease inhibitor (2 μg/ml aprotinine and 2 μg/mlleupeptine) and then lyzed by sonication at 0C. The soluble fraction istaken up by centrifugation at 10,000 revolutions/min (JA21 rotor,Beckman) for 15 minutes at 4° C. and then deposited on a metal-chelatingaffinity column in order to purify the RH24K (Chelating Sepharose FastFlow No. 17057501). The gel was preloaded with Zn²⁺ according to thesuppliers' recommendations and then equilibrated with a sodium phosphatebuffer containing 67 mM NaCl, pH 7.8, and then 200 mM ammonium acetate,0.5 M NaCl, pH 6. The RH24K is eluted with 200 mM ammonium acetate 0.5 MNaCl pH 4. The fractions collected are dialyzed against 50 mM, pH 7.8sodium phosphate buffer.

The purified protein is characterized by electrophoresis on SDS-PAGE andby passage in gel filtration/HPLC. Its purity is greater than 90% andthus comparable with the purity of RH24. When analyzed by massspectrometry, its molecular weight is 28,027, which is compatible withthe theoretical molecular weight 27,992. The protein is recognized bypolyclonal and monoclonal anti-p24 antibodies in Western Blot and inELISA.

EXAMPLE 2 Coupling of RH24K and of RH24 to the NAVE Copolymer

5 μl of polymer solution at a concentration of 1 g/l is added to asolution of protein in a suitable buffer, at a concentration of 1 g/l.

The reaction medium is stirred for 3 hours at 37° C.

The reaction progress is monitored by HPLC, on a Waters steric exclusioncolumn with a 0.1 M, pH 6.8 phosphate buffer as mobile phase.

The coupling yields reported in FIG. 1 show that the modified proteincouples preferably to the polymer and over a wider spectrum ofexperimental conditions.

EXAMPLE 3 Effect of the Nature of the Polymer on the Coupling Yield

The effect of the nature of the comonomer forming part of thecomposition of the polymer is evaluated under the same conditions asthose described above.

The results are given in the table below.

TABLE Coupling yield Polymer (%) MAVE 65 poly(methyl vinyl ether/maleicanhydride PEMA 86 poly (ethylene/maleic anhydride) SMA 49 poly(styrene/maleic anhydride) NVPMA 36 poly (N-vinylpyrrolidone/ maleicanhydride

The effect of the comonomer is quite pronounced since the couplingyields vary within a range between 36 and 86% depending on the nature ofthe comonomer.

EXAMPLE 4 Compared Stability of the Protein/Polymer Conjugate Compoundswith Respect to the Protein Alone

The conjugate compounds were stored at 37° C. in the coupling medium andanalyzed by steric exclusion chromatography in order to evaluate theamount of residual protein in the coupling medium. The results are givenin the graph represented in FIG. 2.

The coupling on polymer thus gives the protein better stability onstorage.

EXAMPLE 5 Directed Biotinylation of RH24K

The conjugate compounds prepared are biotinylated p24s (RH24 or RH24K)which will be used in detection in the p24 antigen sandwich test, inaccordance with the scheme represented in FIG. 3.

The biotin couplings are carried out on RH24K and on RH24, checked andthen analyzed in the antigen sandwich test in order to define thecoupling conditions which are favorable to RH24K.

The proteins are used at a concentration of 1 mg/ml. Different bufferswere tested on the basis of the buffers tested during the p24/MAVEcouplings: 0.1 M borate pH 9.2, 0.1 M Tris pH 7.2, 0.1 M phosphate pH7.2 and 0.1 M carbonate pH 8.5. The following biotin/p24 ratios weretested: 5/1, 10/1, 25/1, 50/1, 100/1. The couplings were incubated for 1hour at 37° C.

The p24/biotin conjugate compounds were used in detection according tothe schema represented in FIG. 3 with monoclonal antibodies and apositive serum.

The RH24/biotin and RH24K/biotin conjugate compounds show activity inthe antigen sandwich test with the monoclonal antibodies and thepositive serum. By way of illustration, FIGS. 4 and 5 show, in graphform, the compared responses of the conjugate compounds obtained withRH24K and RH24 at different degrees of biotin functionalization. FIG. 4relates to the results obtained with a monoclonal antibody, the testbeing performed on a microtitration plate. FIG. 5 represents the resultsobtained on serum with the Vidas immunoanalysis automated machine fromBioMérieux.

RH24K gives a bigger signal than RH24, irrespective of the experimentalconditions tested. An optimum biotin/p24 ratio of 25/1 for themonoclonal antibodies, and of 10/1 for the serum, is observed.

EXAMPLE 6 Influence of the Composition, in Terms of Lysine Residues ofthe Polylysine Tag, on the Coupling Efficacy

The biological material used to evaluate the influence of thecomposition as lysine residues of the polylysine tag on the couplingefficacy is as follows:

the recombinant protein RH24K; this contains, in its amino-terminalpart, a tag [MRGS(H)₆GSVDESM] (SEQ ID NO: 5), which serves to purify itby elation with a metal ion, and, in its carboxy-terminal part, apolylysine tag [PG(K)₆SVDESL] (SEQ ID NO: 4) dedicated to the coupling;this protein can be represented as follows:

MRGS(H)₆GSVDESM-p24-PG(K)₆SVDESL)

the recombinant protein RHK24; this contains, in its amino-terminalpart, a polylysine tag [MRGSCH(K)₂HH(K)₂HH(K)₂GSVDESM] (SEQ ID NO: 6);this protein can be represented as follows:

MRGSCH(K)₂HH(K)₂HH(K)₂GSVDESM-p24

the recombinant protein R24; this contains neither the carboxy-terminalpolylysine tag nor the amino-terminal polyhistidine tag; it can berepresented by p24.

The polymers used are MAVE (maleic anhydride co-methyl vinyl ether). Thecoupling conditions are identical for the three proteins.

Two coupling buffers were used, the 0.1 M, pH 8.2 carbonate buffer andthe 0.05 M, pH 9.0 Tris HCl buffer.

The results are given in the following table illustrating the yield forcoupling the recombinant proteins with the MAVE polymer carried outunder two different reaction conditions. They show the importance of thecontiguity of the lysine residues.

The reason for this is that the coupling yield obtained with the proteinRH24K (6 contiguous lysine residues) is very much greater than thecoupling yield obtained with protein RHK24 (6 lysine residues in threeblocks of 2).

In point of fact, the coupling yield obtained with the protein RHK24 (6lysine residues in three blocks of 2) is similar to that obtained withthe protein R24 (no tag).

TABLE Protein R24 RH24K RHK24 polylysine no PG (K)₆ MRGSCH (K) ₂HH (K)₂tag tag SVDESL HH (K) ₂GSVDESM 0.1 M pH 8.2 30% 95% 33% carbonate buffer0.05 M pH 9.0 35% 100% 20% Tris HCl buffer

EXAMPLE 7 Production of Anti-Polylysine Tag (K)₆SVDESL (SEQ ID NO: 7)Antibodies

The biological material used to obtain the anti-tag antibodies capableof recognizing this tag in the context of a fusion with a recombinantprotein is as follows:

the recombinant protein RH24K; this contains, in its amino-terminalpart, a polyhistidine tag [MRGS(H)₆GSVDESM] (SEQ ID NO: 5), which servesto purify it by chelation with a metal ion, and, in its carboxy-terminalpart, a polylysine tag [PG(K)₆SVDESL] (SEQ ID NO: 4), which is dedicatedto the coupling; this protein is represented by

MRGS(H)₆GSVDESM-p24-PG(K)₆SVDESL

the peptide P400, the sequence of which is C(K)₆SVDESL (SEQ ID NO: 8),coupled to KLH (P400-KLH).

The biological material used to select the antibodies produced is asfollows:

the recombinant protein RH24K

the recombinant protein RH24 containing no carboxy-terminal polylysinetag, and represented by

MRGS(H)₆GSVDESM-p24)

the recombinant protein RH24; this contains neither the carboxy-terminalpolylysine tag nor the amino-terminal polyhistidine tag, and isrepresented by p24.

the peptide P400, the sequence of which is C(K)₆SVDESL (SEQ ID NO: 8).

The following two test formats were used to select the monoclonalantibodies:

a sandwich ELISA test, comprising, in the capture phase, an anti-mousegoat antibody, and, in the detection phase, the antigens RH24K or R24revealed by an anti-24 monoclonal antibody coupled to peroxidase;

an indirect ELISA test, comprising, in the capture phase, the antigensRH24K or R24 or P400, and, in the detection phase, an anti-mouse IgVgoat antibody coupled to peroxidase.

BALB/c and A/J mice were immunized according to the following protocol:3 intra-peritoneal injections with, for the first injection, the proteinRH24K, and, for the following two injections, the peptide 400 coupled toKLH. Fusions were carried out and screenings carried out using thesandwich and indirect ELISA tests described above.

80 hybrids were selected according to the following criteria:

recognition of the peptide P400 (C(K)₆SVDESL) (SEQ ID NO: 8)

recognition of the protein RH24K (MRGS(H)₆GSVDESM-p24-PG(K)₆SVDESL)

non-recognition of the protein RH24 (MRGS(H)₆GSVDESM-p24)

non-recognition of the protein R24.

These antibodies thus recognize the sequence (K)₆SVDESL (SEQ ID NO: 7)fused to a protein, but not the sequence MRGS(H)₆GSVDESM (SEQ ID NO: 5)also containing the SVDESL unit, nor the protein p24.

Five of these hybrids were cloned and produced in the form of ascites:these are the antibodies IG2D4, 2G2B3, 2G4A12, 5F12ES and 14E1G7, all ofIgG1 k isotype.

The results of the sandwich and indirect ELISA tests on the IG2D4antibodies are illustrated in FIGS. 6 and 7. FIG. 6 illustrates asandwich ELISA test and represents the OD as a function of the titer(dilution) of the IG2D4 antibody ascite; the recombinant proteins RH24Kand R24, constituting the detection phase, are used at a concentrationof 0.1 μg/ml. FIG. 7 illustrates an indirect ELISA test and representsthe OD as a function of the titer (dilution)(of the IG2D4 antibodyascite; the recombinant proteins RH24K and R24, constituting the capturephase, are used at a concentration of 0.5 μg/ml and the syntheticpeptide P400 is used at a concentration of 0.05 μg/ml.

Bibliography

(1) Porath J., Carlsson., Olsson., Belfrage J., Nature, 258, 598 (1975)

(2) Porath J., Trends Anal. Chem., 7, 254 (1988)

(3) Cheynet, Protein Expression and Purification, 4, 367-372 (1993)

8 1 38 DNA Artificial Sequence Oligonucleotide used to prepare syntheticadaptor 1 gggaagaaga agaagaagaa gtctgtcgac gaatctct 38 2 42 DNAArtificial Sequence Oligonucleotide used to prepare synthetic adaptor 2ctagagagat tcgtcgacag acttcttctt cttcttcttc cc 42 3 46 DNA ArtificialSequence C-terminal of adaptor 3 ccc ggg aag aag aag aag aag aag tct gtcgac gaa tct ctc tag a 46 Pro Gly Lys Lys Lys Lys Lys Lys Ser Val Asp GluSer Leu 1 5 10 4 14 PRT Artificial Sequence C-terminal of adaptor 4 ProGly Lys Lys Lys Lys Lys Lys Ser Val Asp Glu Ser Leu 1 5 10 5 17 PRTArtificial Sequence Polyhistidine tag 5 Met Arg Gly Ser His His His HisHis His Gly Ser Val Asp Glu Ser 1 5 10 15 Met 6 23 PRT ArtificialSequence Polylysine tag 6 Met Arg Gly Ser Cys His Lys Lys His His LysLys His His Lys Lys 1 5 10 15 Gly Ser Val Asp Glu Ser Met 20 7 12 PRTArtificial Sequence Polylysine tag 7 Lys Lys Lys Lys Lys Lys Ser Val AspGlu Ser Leu 1 5 10 8 13 PRT Artificial Sequence Polylysine tag 8 Cys LysLys Lys Lys Lys Lys Ser Val Asp Glu Ser Leu 1 5 10

What is claimed is:
 1. Method for isolating a target biological materialcontained in a sample, said method comprising: providing a capturephase; placing said target biological material in contact with at leastthe capture phase; and detecting the target biological material bound tothe capture phase, wherein the capture phase comprises: (a) an organicmolecule containing at least one reactive function and (b) at least oneprotein material capable of recognizing or binding, specifically anddirectly or indirectly, to the target biological material, said proteinmaterial containing a specific site for covalent binding to the reactivefunction of the organic molecule, which site is comprised of at leastone tag comprising at least six contiguous residues selected from thegroup consisting of lysine and lysine-based residues.
 2. Methodaccording to claim 1, wherein the capture phase also comprises a label.3. Method according to claim 2, wherein the capture phase is a detectionphase.
 4. Method according to claim 1, wherein a detection phase is alsoprovided, and comprises an organic molecule containing at least onereactive function, at least one protein material capable of recognizingor binding, specifically and directly or indirectly, to the targetbiological material, and a detectable label, said protein materialcontaining a specific site for covalent binding to the reactive functionof the organic molecule.
 5. Method according to claim 4, wherein theorganic molecule and the protein material in the detection phase are,respectively, identical to and/or different from the organic moleculeand the protein material in the capture phase.
 6. Method according toclaim 4, that the organic molecule in the detection phase is the label.7. Method according to claim 1, wherein the tag is placed at the N- orC-terminal end of the protein material.
 8. Method according to claim 1,wherein the reactive function of the organic molecule is selected fromthe group consisting of ester, acid, halocarbonyl, sulfhydryl,disulfide, epoxide, haloalkyl and aldehyde functions.
 9. Methodaccording to any one of claim 1, wherein the protein material comprisesan antigen and the biological material is an antibody that isspecifically recognized by said antigen.
 10. Method according to claim1, wherein the protein material comprises an antibody and the biologicalmaterial is an antigen that is specifically recognized by said antibody.11. Method according to claim 1, wherein the organic molecule is aparticulate or linear polymer.
 12. Method according to claim 11, whereinthe polymer is selected from the group consisting of polylysinehomopolymers, polytyrosine homopolymers, maleic anhydride copolymers,N-vinylpyrrolidone copolymers, natural or synthetic polysaccharides andpolynucleotides, and enzymes.
 13. Method according to claim 12, whereinthe polymer is selected from the group consisting of maleicanhydride/methyl vinyl ether copolymer,N-vinylpyrrolidone/N-acryloxysuccinimide copolymer andpoly-6-aminoglucose.
 14. Method according to claim 1, wherein theorganic molecule is a hapten.
 15. Method according to claim 1, whereinthe organic molecule is bound to a carrier molecule.
 16. Methodaccording to claim 15, wherein the organic molecule is biotin and thecarrier molecule is avidin.
 17. Capture phase for a target biologicalmaterial, comprising: (a) an organic molecule containing at least onereactive function and (b) at least one protein material capable ofrecognizing or of binding, specifically and directly or indirectly, tothe target biological material, said protein material containing aspecific site for covalent binding to the reactive function of theorganic molecule, said site comprising at least one tag comprising atleast six contiguous residues selected from the group consisting oflysine and lysine-based residues.
 18. Capture phase according to claim17, wherein the tag is placed at the N- or C-terminal end of the proteinmaterial.
 19. Capture phase according to claim 17, wherein the reactivefunction of the organic molecule is selected from the group consistingof ester, acid, halocarbonyl, sulfhydryl, disulfide, epoxide, haloalkyland aldehyde functions.
 20. Reagent for isolating a target biologicalmaterial, comprising a capture phase according to claim
 17. 21. Reagentaccording to claim 20, wherein the capture phase is bound, directly orindirectly, to a solid support, by passive adsorption or by covalency.22. Reagent according to claim 20, further comprising a detection phasecomprising: (a) a second organic molecule containing at least onereactive function, and (b) at least one protein material capable ofrecognizing or of binding, specifically and directly or indirectly, tothe target biological material, the protein material of said detectionphase containing a specific site for covalent binding to the reactivefunction of the second organic molecule, this site comprising at leastone tag comprising at least six contiguous residues selected from thegroup consisting of lysine and lysine-based residues.
 23. Capture phaseaccording to claim 17, wherein the organic molecule is a particulate orlinear polymer.
 24. Capture phase according to claim 23, wherein thepolymer is selected from the group consisting of polylysinehomopolymers, polytyrosine homopolymers, maleic anhydride copolymers,N-vinylpyrrolidone copolymers, natural or synthetic polysaccharides andpolynucleotides, and enzymes.
 25. Capture phase according to claim 24,wherein the polymer is selected from the group consisting of maleicanhydride/methyl vinyl ether copolymer,N-vinylpyrrolidone/N-acryloxysuccinimide copolymer, poly-6-aminoglucosehorseradish peroxidase (HRP) and alkaline phosphatase.
 26. Capture phaseaccording to claim 17, wherein the organic molecule is a hapten. 27.Detection phase for a target biological material, comprising: (a) anorganic molecule containing at least one reactive function, (b) at leastone protein material capable of recognizing or of binding, specificallyand directly or indirectly, to the target biological material, saidprotein material containing a specific site for covalent binding to thereactive function of the organic molecule, said site comprising at leastone tag comprising at least six contiguous residues selected from thegroup consisting of lysine and lysine-based residues and (c) adetectable label.
 28. Detection phase according to claim 27,characterized in that the organic molecule is the label.
 29. Detectionphase according to claim 27, wherein the label is selected from thegroup consisting of an enzyme, a protein, a peptide, an antibody, ahapten a fluorescent compound, a radioactive compound, achemiluminescent compound, an electron-density component, a magneticcomponent and analogs thereof.
 30. Detection phase according to claim27, wherein the tag is placed at the N- or C-terminal end of the proteinmaterial.
 31. Detection phase according to claim 27, wherein thereactive function of the organic molecule is selected from the groupconsisting of ester, acid, halocarbonyl, sulfhydryl, disulfide, epoxide,haloalkyl and aldehyde functions.
 32. Reagent for isolating a targetbiological material, comprising a detection phase according to claim 27.33. Reagent according to claim 32, wherein the capture phase is bound,directly or indirectly, to a solid support, by passive adsorption or bycovalency.
 34. Detection phase according to claim 27, wherein theorganic molecule is a particulate or linear polymer.
 35. Detection phaseaccording to claim 34, wherein the polymer is selected from the groupconsisting of polylysine homopolymers, polytyrosine homopolymers, maleicanhydride copolymers, N-vinylpyrrolidone copolymers, natural orsynthetic polysaccharides and polynucleotides, and enzymes. 36.Detection phase according to claim 35, wherein the polymer is selectedfrom the group consisting of maleic anhydride/methyl vinyl ethercopolymer, N-vinylpyrrolidone/N-acryloxysuccinimide copolymer,poly-6-aminoglucose, horseradish peroxidase (HRP) and alkalinephosphatase.